Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-β1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-β1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl(4)-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-β1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-β-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-β1 induction by targeting SMAD4.     

Pinostilbene hydrate suppresses hepatic stellate cell activation via inhibition of miR-17–5p-mediated Wnt/β-catenin pathway

BackgroundIn the development of liver fibrosis, activated hepatic stellate cells (HSCs) contribute to the synthesis and deposition of extracellular matrix (ECM) proteins. HSC activation is considered as a central driver of liver fibrosis. Recently, microRNAs (miRNAs) have been reported to act as key regulators in HSC activation.PurposePinostilbene hydrate (PSH), a methylated derivative of resveratrol, has demonstrated anti-inflammatory, antioxidant and anti-tumour activities. However, the effects of PSH on HSC activation remain unclear.MethodsThe effects of PSH on HSC activation were examined. Moreover, the roles of WNT inhibitory factor 1 (WIF1) and miR-17–5p in the effects of PSH on HSC activation were examined.ResultsPSH induced a significant reduction in HSC proliferation. PSH also effectively inhibited HSC activation, with reduced α-SMA and collagen expression. Notably, it was found that Wnt/β-catenin signalling was involved in the effects of PSH on HSC activation. PSH resulted in Wnt/β-catenin signalling inactivation, with a reduction in TCF activity as well as β-catenin nuclear translocation. Further studies showed that PSH inhibited Wnt/β-catenin signalling via regulation of WIF1 and miR-17–5p. Reduced HSC activation caused by PSH could be restored by loss of WIF1 or miR-17–5p mimics. Luciferase reporter assays further confirmed that WIF1 was a target of miR-17–5p.ConclusionPSH has a significant protective effect against HSC activation. In addition, we demonstrate that PSH enhances WIF1 expression and inhibits Wnt/β-catenin signalling via miR-17–5p, contributing to the suppression of HSC activation.     

Inhibition of miR-188-5p alleviates hepatic fibrosis by significantly reducing the activation and proliferation of HSCs through PTEN/PI3K/AKT pathway

Persistent hepatic damage and chronic inflammation in liver activate the quiescent hepatic stellate cells (HSCs) and cause hepatic fibrosis (HF). Several microRNAs regulate the activation and proliferation of HSCs, thereby playing a critical role in HF progression. Previous studies have reported that miR-188-5p is dysregulated during the process of HF. However, the role of miR-188-5p in HF remains unclear. This study investigated the potential role of miR-188-5p in HSCs and HF. Firstly, we validated the miR-188-5p expression in primary cells isolated from liver of carbon tetrachloride (CCl₄)-induced mice, TGF-β1-induced LX-2 cells, livers from 6-month high-fat diet (HFD)-induced rat and 4-month HFD-induced mice NASH models, and human non-alcoholic fatty liver disease (NAFLD) patients. Furthermore, we used miR-188-5p inhibitors to investigate the therapeutic effects of miR-188-5p inhibition in the HFD + CCl₄ induced in vivo model and the potential role of miR-188-5p in the activation and proliferation of HSCs. This present study reported that miR-188-5p expression is significantly increased in the human NAFLD, HSCs isolated from liver of CCl₄ induced mice, and in vitro and in vivo models of HF. Mimicking the miR-188-5p resulted in the up-regulation of HSC activation and proliferation by directly targeting the phosphatase and tensin homolog (PTEN). Moreover, inhibition of miR-188-5p reduced the activation and proliferation markers of HSCs through PTEN/AKT pathway. Additionally, in vivo inhibition of miR-188-5p suppressed the HF parameters, pro-fibrotic and pro-inflammatory genes, and fibrosis. Collectively, our results uncover the pro-fibrotic role of miR-188-5p. Furthermore, we demonstrated that miR-188-5p inhibition decreases the severity of HF by reducing the activation and proliferation of HSCs through PTEN/AKT pathway.     

Profibrotic effect of miR-33a with Akt activation in hepatic stellate cells

MicroRNAs (miRNAs) attract more attention in the pathophysiology of liver fibrosis and miR-33a has been previously demonstrated as involved in the regulation of cholesterol and lipid metabolism. Transforming growth factor-beta1 (TGF-β1) is generally accepted to be the main stimulating factor in the hepatic stellate cells (HSCs) activation, which plays an important role in hepatic fibrosis. However, the involvement and underlying mechanism of miR-33a and its role in TGF-β1-induced hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-33a in the activation of immortalized human HSCs, Lx-2 cells. Our findings have shown that the expression of miR-33a with its host gene sterol regulatory element-binding protein 2 (SREBP2) was more highly expressed in activation of Lx-2 cells than in quiescent cells. The expression of miR-33a on TGF-β1-induced HSCs activation may be modulated via the activation of PI3K/Akt pathway. In addition, miR-33a significantly correlated with TGF-β1-induced expression of α1 (I) collagen (Col1A1) and α-SMA in HSCs. Bioinformatics analyses predict that peroxisome proliferator activated receptor-alpha (PPAR-α) is the potential target of miR-33a. We further found that anti-miR-33a significantly increases target gene PPAR-α mRNA and protein level, suggesting that miR-33a involved in HSCs function might be modulated by targeting PPAR-α. Finally, our results indicate that the expression of miR-33a increased with the progression of liver fibrosis. These results suggested that anti-miR-33a inhibit activation and extracellular matrix production, at least in part, via the activation of PI3K/Akt pathway and PPAR-α and anti sense of miR-33a may be a novel potential therapeutic approach for treating hepatic fibrosis in the future.     

Bromodomain protein 4 is a key molecular driver of TGFβ1-induced hepatic stellate cell activation

Liver fibrosis is characterized by the excessive deposition of extracellular matrix in liver. Chronic liver injury induces the activation of hepatic stellate cell (HSCs), a key step in liver fibrogenesis. The activated HSC is the primary source of ECM and contributes significantly to liver fibrosis. TGFβ1 is the most potent pro-fibrotic cytokine. Bromodomain protein 4 (BrD4), an epigenetic reader of histone acetylation marks, was crucial for profibrotic gene expression in HSCs. The present study aimed to investigate the roles of BRD4 in TGFβ1-dependent HSC activation and liver fibrosis, focusing on TGFβ1-induced alterations of the levels of the fibrotic-related important proteins in HSCs by employing the heterozygous TGFβ1 knockout mice and BrD4 knockdown in vivo and in vitro. Results revealed that BrD4 protein level was significantly upregulated by TGFβ1 and BrD4 knockdown reduced TGFβ1-induced HSC activation and liver fibrosis. BrD4 was required for the influences of TGFβ1 on PDGFβ receptor and on the pathways of Smad3, Stat3, and Akt. BrD4 also mediated TGFβ1-induced increases in histone acetyltransferase p300, the pivotal pro-inflammatory NFkB p65, and tissue inhibitor of metalloproteinase 1 whereas BrD4 reduced Caspase-3 protein levels in HSCs during liver injury, independent of TGFβ1. Further experiments indicated the interaction between TGFβ1-induced BrD4 and NFkB p65 in HSCs and in liver of TAA-induced liver injury. Human cirrhotic livers were demonstrated a parallel increase in the protein levels of BrD4 and NFkB p65 in HSCs. This study revealed that BrD4 was a key molecular driver of TGFβ1-induced HSC activation and liver fibrosis.     

Extracellular vesicles-derived miR-150-5p secreted by adipose-derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis

Hepatic fibrosis (HF) is involved in aggravated wound-healing response as chronic liver injury. Extracellular vesicles (EVs) carrying microRNA (miR) have been reported as therapeutic targets for liver diseases. In this study, we set out to explore whether adipose-derived mesenchymal stem cells (ADMSCs)-derived EVs containing miR-150-5p affect the progression of HF. Carbon tetrachloride (CCl₄) was firstly used to induce HF mouse models in C57BL/6J mice, and activation of hepatic stellate cells (HSCs) was achieved using transforming growth factor β (TGF-β). EVs were then isolated from ADMSCs and co-cultured with HSCs. The relationship between miR-150-5p and CXCL1 was identified using dual luciferase gene reporter assay. Following loss- and gain-function experimentation, HSC proliferation was examined by MTT assay, and levels of fibrosis-, HSC activation- and apoptosis-related genes were determined in vitro. Additionally, pathological scores, collagen volume fraction ( CVF) as well as levels of inflammation- and hepatic injury-associated genes were determined in in vivo. Down-regulated miR-150-5p and elevated CXCL1 expression levels were detected in HF tissues. ADMSCs-derived EVs transferred miR-150-5p to HSCs. CXCL1 was further verified as the downstream target gene of miR-150-5p. Moreover, ADMSCs-EVs containing miR-150-5p markedly inhibited HSC proliferation and activation in vitro. Meanwhile, in vivo experiments also concurred with the aforementioned results as demonstrated by inhibited CVF, reduced inflammatory factor levels and hepatic injury-associated indicators. Both experiments results were could be reversed by CXCL1 over-expression. Collectively, our findings indicate that ADMSCs-derived EVs containing miR-150-5p attenuate HF by inhibiting the CXCL1 expression.     

PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl₄ and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.     

MFAP2 promotes HSCs activation through FBN1/TGF-β/Smad3 pathway

Liver fibrosis is a chronic inflammatory process characterized by the accumulation of extracellular matrix (ECM), which contributes to cirrhosis and hepatocellular carcinoma. Increasing evidence suggests that the activation of hepatic stellate cells (HSCs) under an inflammatory state leads to the secretion of collagens, which can cause cirrhosis. In this study, we analysed data from the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs) between quiescent and fibrotic HSCs. We found that Microfibril Associated Protein 2 (MFAP2) was elevated in carbon tetrachloride (CCl4)-induced liver fibrosis and Transforming Growth Factor-Beta 1 (TGF-β1)-activated HSCs. Knockdown of MFAP2 inhibited HSC proliferation and partially attenuated TGF-β-stimulated fibrogenesis markers. Bioinformatics analysis revealed that Fibrillin-1 (FBN1) was correlated with MFAP2, and the expression of FBN1 was significantly upregulated after MFAP2 overexpression. Silencing MFAP2 partially attenuated the activation of HSCs by inhibiting HSC proliferation and decreasing collagen deposits. In vitro results showed that the inhibition of MFAP2 alleviated hepatic fibrosis by inhibiting the activation and inducing the apoptosis of active HSCs in a CCl4-induced mouse model. In conclusion, our results suggest that MFAP2 is a potential target for the clinical treatment of liver fibrosis.     

Mechanism of thymosin β4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway

Liver fibrosis is a grievous global challenge, where hepatic stellate cells (HSCs) activation is a paramount step. This study analyzed the mechanism of Tβ4 in ameliorating liver fibrosis via the MAPK/NF-κB pathway. The liver fibrosis mouse models were established via bile duct ligation (BDL) and verified by HE and Masson staining. TGF-β1-induced activated LX-2 cells were employed in vitro experiments. Tβ4 expression was determined using RT-qPCR, HSC activation markers were examined using Western blot analysis, and ROS levels were tested via DCFH-DA kits. Cell proliferation, cycle, and migration were examined by CCK-8, flow cytometry, and Transwell assays, respectively. Effects of Tβ4 on liver fibrosis, HSC activation, ROS production, and HSC growth were analyzed after transfection of constructed Tβ4-overexpressing lentiviral vectors. MAPK/NF-κB-related protein levels were tested using Western blotting and p65 expression in the nucleus was detected through immunofluorescence. Regulation of MAPK/NF-κB pathway in TGF-β1-induced LX-2 cells was explored by adding MAPK activator U-46619 or inhibitor SB203580. Furthermore, its regulating in liver fibrosis was verified by treating BDL mice overexpressing Tβ4 with MAPK inhibitor or activator. Tβ4 was downregulated in BDL mice. Tβ4 overexpression inhibited liver fibrosis. In TGF-β1-induced fibrotic LX-2 cells, Tβ4 was reduced and cell migration and proliferation were enhanced with elevated ROS levels, while Tβ4 overexpression suppressed cell migration and proliferation. Tβ4 overexpression blocked the MAPK/NF-κB pathway activation by reducing ROS production, thus inhibiting liver fibrosis in TGF-β1 induced LX-2 cells and BDL mice. Tβ4 ameliorates liver fibrosis by impeding the MAPK/NF-κB pathway activation.     

Protective effect of Idelalisib on carbon tetrachloride-induced liver fibrosis via microRNA-124-3P/phosphatidylinositol-3-hydroxykinase signalling pathway

Liver fibrosis is the repair process of abnormal connective tissue hyperplasia after liver damage caused by different causes. Inhibition of PI3K/Akt signalling pathway can reduce the deposition of extracellular matrix, inhibit the proliferation of hepatic stellate cells (HSCs), and promote its apoptosis to achieve the purpose of therapy. This study aimed to investigate the effect of Idelalisib (PI3K inhibitor) on carbon tetrachloride (CCl₄)-induced liver fibrosis in mice. We used CCl₄-induced liver fibrosis mouse model in vivo and TGF-β1-stimulated HSCs to evaluate the antifibrosis activity of Idelalisib. In vivo, Idelalisib significantly alleviated CCl₄-induced liver damage, collagen deposition, and hydroxyproline accumulation in mice. Immunohistochemistry and Western blot results showed that Idelalisib could significantly inhibit the expressions of COL1 and α-SMA in a concentration-dependent manner. In cell experiments, Idelalisib significantly inhibited the expressions of COL1, SMA, and p-Smad3 in TGF-β-induced HSCs, thereby inhibiting HSC activation. Flow cytometry and Western blot results showed that Idelalisib significantly promoted TGFβ-induced apoptosis of HSCs after 48 h of administration, but had no significant effect after 24 h. Idelalisib promoted the apoptosis of activated HSCs by inhibiting the PI3K/Akt/FOXO3 signalling pathway. To further explore the mechanism by which Idelalisib inhibited PI3K, we predicted the miRNA targeting PI3K through the database and crossed it with the down-regulated miRNA reported in liver fibrosis mice in the past five years. Finally, we identified miR-124-3p and miR-143-3p. We then demonstrated that Idelalisib significantly promoted miR-124-3p and miR-142-3p in vitro and in vivo. Dual-luciferase report analysis showed that Idelalisib significantly inhibited luciferase activity but had no significant effect on the luc-MUT transfection assay. Finally, we demonstrated that Idelalisib reversed the effects of miR-124-3p inhibitor on the PI3K/Akt/FOXO3 asterisk pathway and caspase-3. Idelalisib has potential as a candidate drug for alleviating liver fibrosis.     

MicroRNA-326 attenuates hepatic stellate cell activation and liver fibrosis by inhibiting TLR4 signaling

Hepatic fibrosis is a chronic inflammatory and reversible repair reaction of the liver under the continuous action of virus or various injuries. In this study, we aimed at identifying the role of miR-326 in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. In this study, the liver fibrosis mouse model was developed by injecting CCl₄. Liver tissue morphology was observed and the expression level of α-smooth muscle actin, collagen1α1 and miR-326 was measured. Target gene identification was performed by loss-of-function and gain-of-function. The effect of miR-326 on the expression level of the cytokines associated with the TLR4/MyD88/nuclear factor-κB (NF-κB) pathway was assessed in vitro and in vivo. We show that miR-326 was downregulated in CCl₄-induced fibrotic mice and activated HSCs. The target gene of miR-326 is TLR4. Moreover, miR-326 inhibited the activation of HSCs in vitro through TLR4/MyD88/NF-κB signaling. miR-326 attenuated hepatic fibrosis and inflammation of CCl₄-induced mice in vivo. Our results demonstrate for the first time that miR-326 inhibits HSC activation through TLR4/MyD88/NF-κB signaling. Furthermore, miR-326 plays critical roles in attenuating liver fibrosis and inflammation, suggesting the therapeutic potential of miRNAs.     

MicroRNA signatures associated with thioacetamide-induced liver fibrosis in mice

Multiple etiologies of liver injury are associated with fibrosis in which the key event is the activation of hepatic stellate cells (HSCs). Although microRNAs (miRNAs) are reportedly involved in fibrogenesis, the complete array of miRNA signatures associated with the disease has yet to be elucidated. Here, deep sequencing analysis revealed that compared to controls, 80 miRNAs were upregulated and 21 miRNAs were downregulated significantly in the thioacetamide (TAA)-induced mouse fibrotic liver. Interestingly, 58 of the upregulated miRNAs were localized to an oncogenic miRNA megacluster upregulated in liver cancer. Differential expression of some of the TAA-responsive miRNAs was confirmed, and their human orthologs were similarly deregulated in TGF-β1-activated HSCs. Moreover, a functional analysis of the experimentally validated high-confidence miRNA targets revealed significant enrichment for the GO terms and KEGG pathways involved in HSC activation and liver fibrogenesis. This is the first comprehensive report of miRNAs profiles during TAA-induced mouse liver fibrosis.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-β(1). Interestingly, TGF-β(1) secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-β(1) restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-β(1)-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-β(1) expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-β(1) and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.